CANCER GENOMICS & PROTEOMICS 9: 357-372 (2012)

Review Genetically Engineered Fusion for Treatment of Cancer

ULRICH H. WEIDLE, BRITTA SCHNEIDER, GUY GEORGES and ULRICH BRINKMANN

Roche Pharma Research and Early Development (pRED), Roche Diagnostics GmbH, Penzberg, Germany

Abstract. In this review, we summarize approaches to treat generated by combining entities derived from the ECDs of cancer with genetically engineered fusion proteins. Such two, or theoretically more, plasma membrane-associated proteins can act as decoy receptors for several ligands or as receptors. The fusion proteins, as outlined, can bind several recruiters of immune effector cells to tumor. Examples of ligands dedicated to one or several receptors inhibiting, interference with -mediated tumor growth and processes such as tumor-associated angiogenesis or human tumor-related angiogenesis with fusion proteins consisting of epidermal (HER)-based signaling. the extracellular domains, and in some cases also of entities Another approach is the recruitment of immune effector cells of one or several receptors and the Fc part of human IgG1, to the tumor with genetically engineered fusion proteins. We are discussed. In addition, we present strategies for discuss fusion proteins of tumor-related antibodies and recruitment of immune effector cells to tumor with fusion cytokines or MHC-class I peptide complexes, T-cell receptor proteins. This can be achieved with fusion proteins consisting (TCR) cytokine fusion proteins, and fusions proteins of a of a tumor-related antibody and a cytokine or major T-cell-recruiting antibody and a tumor-cell receptor ligand or histocompatibilty complex class-I-peptide complexes, by a defined TCR. T-cell receptor cytokine fusion proteins or by combination of a T-cell-recruiting antibody with a tumor-related ligand or a Technologies for Generation of Genetically defined T-cell receptor. Engineered Fusion Proteins

One of the aspects of biologics-based cancer therapy is the Genetically engineered fusion proteins are composed of inhibition of angiogenesis and of tumor-promoting effects of domains of antibodies (predominantly IgG), and of growth factors. Another approach is based on the recruitment additional entities that mediate functionality. The of immune effector cells to the tumor in order to initiate an entities that are derived from antibodies can either contain antitumor immune response. These approaches are based on binding regions or constant antibody regions. the identification of the hallmarks of cancer (1-3). Using the Binding region modules are applied for targeting first approach, several recombinant receptor decoys are under approaches, i.e. to direct a fused ‘effector’ payload pre-clinical and clinical investigation. They are based on specifically to those cells that shall be addressed. These genetic fusion of the extracellular domain (ECD) of a binding entities are composed, in most cases, of variable receptor and the Fc-part of human immunoglobulin IgG1, regions (Fv) of antibodies which specifically bind the desired which improves the pharmacokinetic properties of the fusion antigen. Fv fragments are heterodimers of variable heavy protein. Chimeric decoy receptor fusion proteins can be chain (VH) and variable light chain (VL) domains. They are the smallest modules that retain a complete structure of the binding region of antibodies. Their small size (approx. This article is freely accessible online. 25 kDa) enables the generation of small fusion proteins which may penetrate tissues and tumors quite rapidly. Such Correspondence to: Ulrich H. Weidle, Roche Pharma Research and molecules may be particularly useful in applications that Early Development (pRED), Roche Diagnostics GmbH, Im require rapid tissue/tumor penetration, e.g. for targeted cancer Nonnenwald 2, D-82372 Penzberg, Germany. E-mail: therapy. The complete removal of the constant regions of [email protected] antibodies not only reduces the size of fusion proteins, but it Key Words: Angiogenesis, antibody cytokine fusion proteins, also restricts the functionality of antibody modules to chimeric extracellular receptor domains, decoy receptor, HER ‘targeting-only’. Removal of the constant domains eliminates signaling; Fc-based fusion proteins, immune effector cell any (Fc-receptor mediated) effector functionality of the parent recruitment, treatment resistance, review. antibody, such as complement-dependent cytotoxicity (CDC)

1109-6535/2012 $2.00+.40 357 CANCER GENOMICS & PROTEOMICS 9: 357-372 (2012) or antibody-dependent cellular cytotoxicity (ADCC). the modulation of size and the pharmacokinetics of the Application of Fvs without attached constant domains therapeutic fusion proteins. The Fc-fragment of antibodies generates technical challenges, in particular stability issues: (IgG) starts with an N-terminal at the flexible hinge region of while fragment antigen binding (Fabs), [heterodimers antibodies, thereafter followed by CH2 and CH3 domains. composed of VH first constant domain of the heavy chain These entities form stable homodimers, covalently linked by (CH1) and VL (CL)] are by themselves quite stable interchain disulfide bonds between cysteine residues in their heterodimers, VH-VL heterodimers are instable. The CL and hinge regions. The fusion partners of Fc-containing therapeutic CH1 domains contribute significantly to the formation of the proteins are connected to each other in a combinatorial heterodimeric Fabs, and their interactions (including an manner. In most cases, the fusion to Fc is made at the hinge interchain disulfide at the C-terminal end of CH1 and CL) region, followed thereafter by CH1 and CH2. Hinge regions stabilize Fab fragments. Once these domains are removed, are flexible by nature, thus fusion at this position minimizes VH and VL have only very limited capability to form or prevents undesired interactions and steric hindrance. This functional Fv heterodimers. Furthermore, VH-VL interfaces combinatorial composition retains the functionality of the Fc are too weak to stably retain heterodimeric Fvs without CH1 region, as well as that of the additional entity, as will be shown and CL attached (4, 5). One method that has frequently and in this review. Constant regions bind Fc receptors, including successfully been applied to overcome the problem of instable FcRn (12, 13). Binding to FcRn, which enables antibody VH-VL heterodimers is the covalent connection of VH and recycling/export from cells, is the key contributor to the long VL by a flexible peptide linker. These linkers are frequently serum half-life of antibodies. This favorable pharmacokinetic composed of glycine and serine, containing stretches of 15- property can also be transferred to fusion proteins that contain 20 amino acids. The tethering between VH and VL generates Fc domains as FcRn-binding modules. A further advantage of single-chain Fv molecules (6, 7), which are more stable having Fc regions as part of recombinant fusion proteins is compared to VH-VL heterodimers by themselves. Single- that they can be applied to facilitate production and chain (sc)Fvs are widely applied as recombinant binding downstream processing. For this reason, Fc fusions can be modules and components of fusion proteins. expressed and purified by procedures that are similar to those Additional technologies have been applied to further applied for the production of therapeutic antibodies. This stabilize Fv fragments for therapeutic applications. For some includes expression in mammalian cells, secretion into culture variable regions, linkage of VH and VL by flexible peptide supernatants and subsequent affinity-based (ProteinA) linkers only partially restores its stability. Such scFvs still purification steps. Examples of genetically engineered fusion have a tendency to dissociate and in consequence have a proteins that contain constant regions of antibodies are ‘ligand propensity to aggregate. These problems can be overcome by trap’ molecules such as those containing extracellular domains increasing the stability of the VH-VL interface by mutations of (FGF), and/or by introduction of covalent disulfide connections receptor (EGFR), human epidermal growth factor receptor-2 between the VH and VL domains (8-10). The fusion partners (HER2), and of vascular endothelial growth factor (VEGF) of Fv-containing proteins are connected to each other in a receptors (Figure 2, A-F). modular manner. In most cases, flexible linker or connector sequences are set between the Fv module and the additional VEGF Trap entity to prevent undesired interactions (including steric hindrance) between the fusion partners. This modular The members of VEGF family are involved in processes such composition retains the binding specificity of the Fv region as angiogenesis, lymphangiogenesis, vasculogenesis, and are as well as the functionality of the targeted-effector entity. also modulators of host innate and adoptive immunity (14). Engineered Fv fusion proteins can be efficiently produced in Clinical studies have validated VEGF as a target for treatment bacteria such as Escherichia coli, either as soluble proteins of cancer and vascular diseases affecting the eye. These studies or as inclusion bodies, followed by re-folding to obtain fully have led to the approval of drugs targeting the VEGF pathway, active proteins (11). Examples for genetically engineered such as an antibodies directed against VEGF-A (Avastin), and fusion proteins that contain variable antibody domains as cell kinase inhibitors which inhibit activation of the respective targeting modules are the cytokine fusions that contain receptors (VEGFRs) (15, 16). The biological functions of -2 (IL2), or tumor necrosis factor-α (TNFα), or VEGFs (A, B, C, D and E) are mediated by interactions with those that display human leukocyte antigen (HLA) peptide VEGFRs 1, 2 and 3. VEGFRs 1 and 2, each contain seven complexes (Figure 1, A-D). different ECDs. A soluble VEGF trap was created by genetic Constant region entities of antibodies are also applied for fusion of domain 2 of VEGFR1 to domain 3 of VEGFR2 with the generation of fusion proteins. In contrast to antibody- the Fc portion of human IgG1, resulting in a composite soluble binding entities (for specific targeting approaches), the main decoy receptor referred to as (17) (Figure 2F and rationale for using constant region entities as fusion partners is 3D). It binds to all VEGF-A isoforms generated by differential

358 Weidle et al: Genetically Engineered Fusion Proteins (Review)

Figure 1. Design and composition of fusion proteins that combine antibody-derived targeting domains with effector modules. The domain compositions of fusion proteins are shown schematically in the upper panels and the sequences that encode these entities and summarized in lower panels of each figure. The general design principle includes recombinant fusion of cell surface, targeting modules (blue) to effector modules (yellow). Flexible linker or connector sequences are applied to combine variable heavy-chain (VH) and variable light-chain (VL) domains, and are also incorporated at the fusion sites of Fvs to effector entities (red). These linker and connector sequence stretches contain in many cases serine and glycine residues to permit flexibility. A and B: Monomeric and di-meric fusion proteins for targeted accumulation of IL2. C: Targeted tri-meric tumor necrosis factor. D: Molecules that are applied for targeted accumulation of human leukocyte antigen (HLA)-presented peptides.

splicing, such as VEGF121, VEGF165, VEGF189 and Trap forms an inert 1:1 complex with VEGF that remains in VEGF206, in addition to other members of the VEGF family the circulation and can be monitored, thus providing a such as VEGF B, C and D, as well as biomarker for predicting efficacious angiogenic blockade (23). (PLGF), which is also involved in pathological angiogenesis Measurement of the level of free and bound VEGF provides an (15). The binding to all isoforms of VEGF-A occurs with high indicator of the doses of the VEGF Trap required for capturing affinity (Kd <0.5 pM) and the Kd for hPLGF is in the range of all available VEGF. VEGF Trap exhibits excellent 25 pM. Several pre-clinical properties are attributed to VEGF pharmacological properties and has a circulation half-life of 2- Trap, such as normalization of the tumor vasculature, pruning 3 weeks, allowing for bi-weekly or even less frequent dosing of excess vessels, reduction of interstitial fluid pressure and (23). It should be kept in mind that trapping all members of the vascular permeability (18-20). VEGF Trap has shown VEGF family at first glimpse seems to be a conceptual significant antitumoral and antiangiogenic activity in numerous advantage in comparison to Avastin which binds exclusively to pre-clinical models (21). A significant impact of the VEGF VEGF-A. However, it has been shown that VEGF-B and Trap was documented when administered together with VEGF-D can have a negative impact on angiogenesis and chemotherapy or radiation (21). Similarly to Avastin, the VEGF lymphangiogenesis (24, 25) and in this context controversial Trap has a marked effect on pre-existing or newly-generated effects for PLGF have emerged (26). blood vessels (22). While antibodies form multimeric Aflibercept is currently clinically-evaluated in three phase complexes that are readily cleared from the circulation, VEGF III studies in combination with chemotherapy for second-line

359 CANCER GENOMICS & PROTEOMICS 9: 357-372 (2012) metastatic colorectal cancer (mCRC), second-line non-small In order to sequester mitogenic and angiogenic FGFs, FP- cell lung carcinoma (NSCLC) and first-line hormone- 1039, a fusion protein consisting of the ECD of FGFR1 IIIc refractory prostate cancer (27). Aflibercept was approved for and the Fc domain of human IgG1 was experimentally wet macular degeneration in the US, and phase III studies evaluated (41) (Figure 2A and 3A). Surface plasmon- are currently being conducted as a first-line treatment in resonance experiments indicated binding to FGF1, FGF2, combination with docetaxel and prednisone in patients with FGF4 and FGF18 (41). FP-1039 reduced the number of viable androgen-resistant prostate cancer and in patients with Caki-1 and A549 tumor cells in a concentration-dependent mCRC after failure of an oxaliplatin-based regimen together manner and displayed potent anti-angiogenic activity in FGF2- with folinic acid plus fluoruracil and irinotecan (FOLFIRI). and VEGF-induced angiogenesis in matrigel plugs in mice (41). FP-1039 elicited dose-dependent inhibition of growth of Fibroblast Growth Factor Receptor 1 (FGFR1) Fc the head and neek squamous cell carcinoma (HNSCC) cell lines 584-A2, CCL30 and Detroit 562, but not of UMSCC19, The FGFs are a family of proteins comprising of 22 which produces small amounts of FGF2 (42). FP-1039 structurally related members with a wide range of biological significantly inhibited tumor growth of a renal carcinoma activities (28). FGF1 and FGF2 were the first angiogenesis xenograft model (Caki1) in a dose-dependent manner. Decrease factors identified (29, 30). FGFs activate a family of tyrosine of tumor vascularization and inhibition of phosphorylation of kinase receptors, designated as FGFR1-FGFR4 (31, 32). They ERK1/2 was observed. Tumor growth of primary human tumor are composed of an ECD with three Ig-like domains (D1, D2, xenografts was also inhibited (43). Single-agent therapeutic D3), a transmembrane domain and a cytoplasmic tyrosine efficiency was observed in vitro and in vivo with MFE-280 kinase domain.The ligand binding site is located in the Ig-like endometrial carcinoma cells which carry the activating S252W domains D2 and D3 and the amino acids connecting them FGFR1 mutation, whereas HEC-1B cells with wild-type (33). FGFRs can be spliced differentially, FGF binding FGFR1 status were much less sensitive (43). Phase I studies specificity is determined by the third Ig-like domain, the N- are currently being performed for this rare population of terminal half of which by an invariant exon IIIa, with patients with endometrial carcinoma (43). alternative usage of IIIb and IIIc exons for the C-terminal half. The paracrine FGFs bind to their receptors together with FGFRs containing exon IIIb are expressed on mesenchymal heparin sulfate proteoglycan (HSPG), resulting in cells and those with FGFR IIIc are expressed on epithelial dimerization and activation. The mode of action of FP 1039 cells, establishing a paracrine signaling relationship between is not yet clearly resolved since paracrine FGFs can be pre- epithelial and mesenchymal tissues. FGFR IIIb binds bound to HSPG on the cell surface (28) rather than being efficiently to FGF1, FGF3 and FGF10, whereas FGFRIIIc present in the circulation. FGFRs have also significant binds to FGF1, FGF2, FGF6, FGF8 and FGF9 (34). The role affinity for HSPG and therefore FP-1039 could also be of FGF-FGFR interactions in the development of vasculature directed to the extracellular matrix. Therefore, competition and tumor angiogenesis has been extensively documented (35, of FP-1039 with the binding of FGFs to HSPGs might be 36). An autocrine growth factor role for FGF2, FGF9 and part of the underlying mode of action. FGFR1, FGFR2 has been reported (37). Selective amplification of the FGFR1 in lung squamous cell Activin-like Receptor Kinase 1 (ALK1) Fc carcinomas and cell lines emphasizes the role of FGF FGFR signaling in these tumors (38). Amplification of FGFR2 is ALK1 is a type I transforming growth factor β (TGFβ) observed in 4-20% of patients with breast cancer, while receptor which is preferentially expressed in proliferating FGFR4 is overexpressed in 30% of patients (38). Ligand- vascular endothelial cells (44, 45). Upon ligand binding, the dependent mutations of FGFR2 (S252W/P253R) are observed type II serine-threonine kinase receptor phosphorylates and in 7% of endometrial carcinomas (39, 40). activates ALK1, which by itself phosphorylates a SMAD

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Figure 2. Design and composition of Fc fusion proteins. The domain compositions of Fc fusion proteins are schematically shown in the upper panels and the sequences that encode these entities are summarized in lower panels of each figure. The general principle for design of Fc fusions includes recombinant fusion of effector domains (e.g. extracellular domains of receptor tyrosine kinases or peptides that capture ligands (blue or green) at a hinge region of IgGs (red) to constant regions (CH2 and CH3, grey). These molecules form dimers via their dimerization propensities of CH2-CH3 domains. These dimers are stabilized by disulphide bonds in their hinge regions. A: fibroblast growth factor (FGF) Trap; B: human epidermal growth factor receptor (HER) Trap; C: angiopoeitin (Ang) Trap; D: domain structure of vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2 and receptor in endothelial cells 2 (TIE2); E: double antiangiogenic protein (DAAP); F: vascular endothelial growth factor (VEGF) Trap.

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361 CANCER GENOMICS & PROTEOMICS 9: 357-372 (2012) protein (SMAD 1/5/8). Phosphorylated SMADs dimerize and FGF and BMP10 induced vessel formation. ALK1 Fc with SMAD-4 and the complexes translocate into the nucleus treatment reduced tumor burden in mice receiving orthotopic and mediate transcription of involved in endothelial grafts of MCF-7 mammary adenocarcinoma cells, indicating cell function and angiogenesis (46). Involvement of ALK1 that ALK1-Fc has a role in mitigating vessel formation. in lymphangiogenesis has been also demonstrated (47). Making use of RAP-041, it was shown that blunting of TGFβ, bone morphogenetic protein 9 (BMP9) and BMP10 ALK1 signaling retards the angiogenic switch and tumor have been identified as ligands of the type II receptor (48) growth in the transgenic Rip-1-Tag2 mouse model of and endoglin, a co-receptor for TGFβ, displays a modulatory endocrine pancreatic tumorigenesis (53) in which BMP9 and function for the ALK receptor complex (46). Genetic studies TGFβ are increased during the tumor progression pathway. of ALK1–/– mice and zebrafish with loss-of-function RAP-041 impairs tumor angiogenesis in vivo. It was mutations indicated a role for ALK1 in vessel maturation, demonstrated that ALK1 is a haploinsufficient gene in the differentiation of pericytes and in the organization and context of tumor angiogenesis since ALK1+/– Rip1/Tag2 patency of neo-angiogenic vessels (49, 50). In humans, type mice displayed a reduction in the number of angiogenic 2 hereditary hemorrhagic telangiectasia (HHT2), which islands, as well as a reduced total tumor burden (54). exhibits direct arterovenous connections without an In humans, ALK1 is expressed in a broad range of tumor intervening capillary bed, is linked to inactivating mutations types (46). Acceleron Pharma has started a phase I study of of ALK1 (51). VEGF and basic fibroblast growth factor the ALK1 Fc fusion protein ACE-041 in patients with (bFGF) can promote endothelial cell attachment, spreading advanced solid tumors or refractory multiple myeloma, and tubulogenesis in an ALK1-dependent manner (52). Since assessing safety and tolerability of ACE-041, as well as ALK1 cannot bind to VEGF or bFGF, these findings point changes in tumor metabolism by 18F-deoxyglucose positron to a possible cross-talk between these signaling systems. emission tomography (46). The side-effect profile arising due In order to elucidate the biological function of ALK1 in to dual inhibition of BMP9 and BMP10 will be monitored several experimental systems, ALK1 ligand traps have been carefully, since it has been shown that BMP10 ablation in constructed (53, 54). Murine Alk1 ECD has been fused to mice leads to embryonic lethality due to impaired cardiac mouse Fc (RAP- 041), and in huALK1 Fc (ACE-041), the growth and function (46). Taken together, the pre-clinical corresponding human equivalents have been fused. The data available, point to a role of attenuation of ALK1 murine Alk1 ligands have been shown to bind to human signaling of this decoy receptor in mouse models of cancer ALK1 and vice versa (53). Surface plasmon resonance for inhibition of tumor angiogenesis and growth. experiments indicated binding of BMP9 and BMP10 with high affinity and not TGFβ by the chimeric fusion proteins Podoplanin Fc (53, 54). ALK1 Fc blocks BMP9 signaling in human umbilical venule endothelial cells (HUVECs) and reduces Podoplanin is a transmembrane glycoprotein that is abundantly the expression of transcription factor, encoded by the ID1 expressed on lymphatic vessels but not on blood vascular gene (Id-1), induces VEGF expression and represses the endothelial cells in vitro and in vivo (55, 56). Podoplanin seems antiangiogenic factor thrombospondin-1. In a chick to be involved in cytoskeletal organisation because it interacts chorioallantoic membrane assay, ALK1 Fc reduced VEGF, with proteins of the ezrin/radixin/moesin family, which function

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Figure 3. Structure models of cytotoxic fusion proteins. The structure models were generated based on available structure data of domains or entities. Structural data from the Protein DataBank (PDB, Nov. 2011) (134) were assembled and minimized using DiscoveryStudio31 (135). Ribbon representations are shown to facilitate identification of secondary structures where the N-terminal domain is placed on the top and the C terminal domain on the bottom. The color scheme of the models follows that of Figure 1 and 2. A: Model of the of fibroblast growth factor receptor 1 (FGFR1) Trap composed of several different structures starting with the nuclear magnetic resonance (NMR) structure of domain 1 (pdbcode: 2CKN), a model of the acidic box based on the homology to part of coagulation factor VII, in orange (pdcode: 3CDZ), followed by domains 2 and 3, in blue (pdcode: 3OJV), and, at the C-terminal end, the Fc portion, in grey (pdbcode: 1HZH). B: The HER Trap model is composed of the extracellular domain (ECD) domain of epidermal growth factor receptor (EGFR), in blue (pdbcode: 3NJP) and the ECD domain of HER3, in green (pdbcode: 1M6B), fused to the Fc portion, in grey (pdbcode: 1HZH). C: Double antiangiogenic protein (DAAP) model is based on the first two immunoglobulin domains and the three EGF domains of tyrosine kinase receptor in endothelial cells 2 (TIE2), in green (pdbcode: 2GY7), domain-2 of vascular endothelial growth factor receptor-1 (VEGFR1), in blue (pdbcode: 2XAC), and the Fc portion, in grey (pdbcode: 1HZH). D: Model of VEGF Trap (Aflibercept) is based on the crystal structure of VEGFR1 domain-2, in dark blue (pdbcode: 1QSZ), followed by domain-3 of VEGFR2, in light blue (pdcode: 2X1W), and fused to the IgG1 Fc portion, in grey (pdbcode: 1HZH). E: A major histocompatibility complex (MHC) complex structure comprising the α chain of the MHC allele 201, in yellow, the common β2-microglobulin domain, in green, and a peptide in the groove, in magenta (pdbcode: 2VLR), is used to build the model of a fusion with on scFv, in light and dark blue; the linkers comprising G4S motifs are indicated in red.

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363 CANCER GENOMICS & PROTEOMICS 9: 357-372 (2012) as cross-linkers between actin filaments and the plasma cytokines and angiogenic myeloid cells thus preparing the membrane (57). Podoplanin-null mice die at birth of respiratory vasculature for a proliferative response to angiogenic growth failure and display numerous lymphatic defects, indicating that factors (67). Angpt2 knock-out mice are born normally, but podoplanin is essential for the function and development of have defects in lymphatic vasculature, have edema, and the lymphatic vessels (58, 59). The function of the podoplanin remodeling of the retinal vessels is impaired and many of ectodomain remains to be investigated in more detail. them die at postnatal day 14 (67). With Angpt2 knock-out Interactions with the following proteins have been documented: mice, it was shown that host-derived Angpt2 affects early galectin 8, a modulator of several functions of lymphatic steps of tumor development and vessel formation, but is endothelial cells (60); C-type lectin-like receptor (CLEC)-2 on dispensable for later stages (68). the surface on platelets involved in mediating podoplanin- The functional contribution of the ANGPT-TIE2 system in induced platelet aggregation (61); and the lymphatic-specific angiogenesis and tumor growth was assessed with AMG 386 chemokine (C-C motif) ligand-21 (CCL21), a chemoattractant [2xCon4(c)], a peptide Fc fusion protein (peptibody) which for chemokine (C-C motif) receptor-7 (CCR7), expressed on prevents the interaction of ANGPT1 and ANGPT2 with their subsets of immune cells (62). To explore the function of receptor TIE2 (69). Phage-display libraries were planned podoplanin in more detail, podoplanin Fc was evaluated in against human ANGPT2 , converted into peptide-Fc fusions several experimental systems. It was shown that podoplanin Fc and expressed in Escherichia coli. AMG 386 potently inhibits adhesion, migration and tube formation of lymphatic neutralizes human ANGPT2/TIE2 interaction (IC50=23 pM), endothelial cells, as well as lymphatic vessel formation, in the as well as human ANGPT1/TIE2 interaction (IC50=900 pM), mouse embroid body assay and impairs lymphatic vessel with similar potency for murine and rhesus monkey Angpt2. formation in inflamed mouse cornea in vivo (63). Podoplanin AMG 368 is an Fc-fusion protein with 58 amino acids fused Fc inhibited skin tumor formation in a transgenic skin cancer at its C-terminus (Figure 2C). model, but also induces intravascular coagulation. This property Systemically administered AMG 386 induced regression of is independent of the Fc portion of the molecule and it was large A431 (human epidermoid tumor) and Colo 205 (human shown that MCF-7 cells expressing transmembrane podoplanin colorectal carcinoma) xenografts, with documented on their surface were able to accelerate coagulation of mouse suppression of proliferation of endothelial cells in vivo, blood (64, 65). If the platelet aggregation activity of podoplanin whereas it had no impact on proliferation of these cells in can be eliminated, e.g. by mutation of the crucial threonine 52 vitro. AMG 386 also prevented neovascularization in a rat (63), podoplanin Fc might become an agent interfering with corneal model of angiogenesis. A pharmacological effect on lymphangiogenesis in a therapeutic setting. Interestingly, tumor epiphyseal growth plate thickness was observed in rats treated lymphangiogenesis and metastasis to lymph nodes are also with AMG 386 (69). Epiphyseal thickness is considered to be induced by cancer cell-based expression of podoplanin (65). a consequence of anti-angiogenic therapy in growing rodents Endothelin-1 (ET-1) might play a crucial role in this context treated with VEGF antibodies (70). AMG 368 selectively since it was found that ET 1 expression is increased in inhibits ANGPT2, but also displays some inhibitory activity podoplanin-1 overexpressing tumor xenografts. ET-1 can against potentially antiangiogenic ANGPT1. Despite this interact with endothelin receptor B (ENDRB) on lymphatic ANGPT1 neutralizing activity, AMG 368 elicits a similar endothelium and might induce lymphangiogenesis (65). response profile to the more ANGPT2 selective agents L1-7 (N) and Ab 536 (69). This may indicate that ANGPT2 might Trap be the dominant angiogenic factor and ANGPT1 has a limited function in the context of the experimental settings as The human angiopoietin (ANGPT) tyrosine kinase receptor described above. In order to elucidate the consequences of in endothelial cell (TIE) systems consists of two tyrosine ANGPT1 inhibition, ANGPT1-specific inhibitory molecules kinase receptors (TIE1 and TIE2) and three secreted ligands need to be generated. AMG 386 is currently being evaluated in (ANGPT1, ANGPT2 and ANGPT3) (66, 67). ANGPT1 phase II studies in a variety of cancer types, such as HER2- functions as a TIE2 receptor agonist, whereas ANGPT2 negative breast cancer, hepatocellular, ovarian, colorectal, normally acts as an ANGPT1 antagonist. ANGPT1 can gastric and renal cancer (71-73). As the safety profiles of excert an antiangiogenic function, whereas ANGPT2 AMG 386 and chemotherapy do not overlap considerably, mediates a pro-angiogenic function, and the physiological AMG 386 may be combined with chemotherapy. effects of ANGPT3 are poorly resolved (66, 67). ANGPT1 is constitutively expressed in many human tissues, whereas ANG VEGF Trap ANGPT2 is predominantly expressed in tissues undergoing vascular remodeling. ANGPT2 can promote the dissociation A chimeric decoy receptor, double antiangiogenic protein of pericytes from pre-existing vessels, increase vascular (DAAP), which can simultaneously bind VEGF-A and permeability and facilitate the infiltration of proteases, and blocking their actions was designed (74)

364 Weidle et al: Genetically Engineered Fusion Proteins (Review)

(Figure 2D, E and 3C). Several reports have indicated that The other two receptors form heterodimeric signaling ANGPT-1 and ANGPT-2 are up-regulated in the context of complexes with other ligand-bound HER family members angiogenic rescue when VEGF-A/VEGFR2 signaling is (80). Seven ligands were shown to bind to EGFR/HER1: EGF, blocked (75-78). heparin-binding EGF (HB-EGF), TGFα, (AR), DAAP is composed of the two Ig-like domains and the (BTC), (EPG) and (EPR). Four three EGF-like domains (E1, 2 and 3) of TIE-2, the second (NRGs) have been identified. NRG1, NRG2, IgG domain of VEGFR1 and the Fc portion of IgG1. It was HRG1 and HRG2 bind to HER3 and NRG1 to 4 bind to shown that DAAP binds to hVEGF-A165, mouse (m) Vegf- HER4. In addition, BTC, HB-EGF and EPR can bind to A164, hVEGF-A189, mPLgf, hANGPT2, hANGPT1, HER4 (81). The seven ligands which bind to the EGFR are mAngpt3 and hANGPT4, but not to hVEGF-C, hVEGF-E, synthesized as transmembrane precursors, cleaved by human angiopoietin-like-2 (hANGTPL-2) or mAngptl-4. It metalloproteases thus releasing mature growth factors (82, 83). should be stressed-out that ANGPT1 has been described as HER signaling is dysregulated in many types of cancers an antiangiogenic factor, and ANGPT2 as a pro-angiogenic and based on this observation, drugs for treatment of cancer factor, whereas the function of ANGPT4 has been poorly have been developed, such as , Herceptin, resolved (66, 67). For comparison, VEGF Trap was able to and -DM1 (TDM1), as well as bind to all isoforms of VEGF-A but not any of the small-molecule kinase inhibitors, such as , angiopoietins, whereas TIE2 Fc was able to bind to all and (84, 85). A common theme for these mono- or ANGPTs tested, but not to VEGF. In vivo evaluation of dual-specific target inhibitors is the development of DAAP in an established Lewis lung carcinoma (LLC) tumor resistance during treatment (86, 87), with involvement of model demonstrated regressions of VEGF-A induced active HER family members which are not targeted by the specific vessel sprouting and network formation, but also blockage of drug. Therefore, an approach targeting several members of ANGPT-induced vessel enlargement in tumor angiogenesis. the ERBB family and their ligands might be superior from a DAAP was shown to inhibit growth and metastasis of conceptual point of view. implanted B16 melanoma cells and its combination with Based on this reasoning an HER-ligand binding protein dacarbazine or cisplatin in the LLC model resulted in has been designed. RB200 (Hermodulin) is an HER1-HER3 improved efficacy in comparison to treatment with the single- heterodimer dimerized with an Fc fragment of human IgG1 agents. Overall, the in vivo experiments indicate superiority (88-91) (Figure 2B and 3B). Binding studies revealed that of DAAP in comparison to treatment with VEGF Trap and RB200 interacts with HER1 ligands such as EGF, TGFα and TIE2-Fc and the combination of the latter agents (74). DAAP HB-EGF, as well as HER3 ligands NRG1 and HRG, and also reduced growth and metastasis of orthotopically inhibits EGF and NRG1 stimulated HER family protein implanted CT-26 colon cancer and in the transgenic MMTV- phosphorylation. RB200 was shown to inhibit both growth- PyMT breast cancer model. In an ovarian orthotopic cancer factor stimulated and unstimulated cell proliferation in model (MDAH-2774), reduction of vascular leakage and monolayer cultures and synergistic growth inhibition was induction of tumor vessel normalization was shown. A close observed in combination with tyrosine kinase inhibitors correlation between in vivo efficacy and expression of VEGF- (88). Single-amino acids were identified in the HER1 and A and ANGPT-2 in the models referred above was noted (74). HER3 part of the ligand binding trap which confer high Thus, simultaneous inhibition of VEGF-A and ANGPTs affinity sequestration of the cognate ligands (91). A single seems to be an effective strategy in blocking angiogenesis, mutation (T15S, EGFR subdomain I) increased affinity for metastasis, vascular leakage and potential treatment of VEGF- EGF two-fold, TGFα 26-fold and HB-EGF six-fold. treatment-resistant tumors. Whether the potential dual Another mutation (Y246A, HER3 subdomain II) enhanced function of ANGPT1 and ANGPT2, with respect to inhibition NRG1 binding eight-fold, probably by interfering with and promotion of tumor-related angiogenesis, is a critical subdomain II-IV interactions. Further work revealed that the issue for safety and clinical efficacy remains to be resolved. HER3 subunit of an HER1/HER3 heterodimer suppresses EGFR ligand binding. Reverting this suppression by HER Trap mutation resulted in enhanced ligand binding (EGF 10 fold, TGFα 34 fold, HB-EGF seven-fold, NRG1 31-fold). The (RTK) family of the HER Increased improvement of ligand-binding was reflected in (ERBB) or EGFRs comprises four members: EGFR/HER1, improved inhibition of tumor cell proliferation (91). RB200 HER2, HER3 and HER4 (79). When bound to a ligand they inhibited xenograft growth of A431 (epidermal carcinoma) undergo dimerization and transmit intracellular signals and H1437 (NSCLC) (91). Further experiments need to be resulting in cell migration, proliferation and differentiation. performed in order to demonstrate differentiation of this Two of these receptors, HER1 and HER4 are autonomous, HER-ligand trap from other HER-signaling inhibitors and in undergoing dimerization and signaling upon ligand binding. treatment-resistant settings.

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Transmembrane- and Calcium- evaluation in a therapeutic setting, some of these fusion modulating Cyclophilin Ligand proteins were also evaluated in an adjuvant setting. For (CAML) Interactor (TACI) Fc example, targeted IL2 was shown to enhance immune responses induced by DNA vaccination (108). Antibodies B-lymphocyte stimulator (BLyS) (also known as BAFF, directed against CD20, epithelial cellular adhesion molecule TALL-1, THANK and zTNF4) is a member of the tumor (EpCAM), ganglioside GD2 and single-stranded DNA in necrosis factor (TNF) family and is functionally involved in conjunction with IL2, IL12, GM-CSF and TNFα have been survival of B-lymphocytes, regulation of peripheral B-cell evaluated pre-clinically (106, 107). The side-effect profile of populations and is overexpressed in several B-cell these fusion proteins is triggered by the tumor specificity of malignancies. BLyS binds to three receptors: B-cell maturation the antibody moiety. The fibronectin B-domain, generated by antigen (BCMA), TACI and B-cell activating factor of the differential splicing, was detected selectively in the tumor necrosis factor family receptor (BAFF-R) (92, 93). A extracellular matrix of newly formed tumor-related blood proliferation-inducing ligand (APRIL), a factor closely related vessels. An antibody (L19) with specificity for this domain to BLyS in sequence and function and which is also was identified (109). Fusion proteins of this antibody with overexpressed in several B-cell malignancies, has also been TNFα, IL2 and IL12 have been evaluated pre-clinically (110). identified (92). TACI and BCMA bind to BLyS and APRIL A fusion protein between L19 and IL2 gave rise to an and BAFF-R binds to BLyS. This indicates that BLyS can improved therapeutic index for IL2, significant antitumor signal through three receptors, whereas APRIL signals through activity, and infiltration of experimental tumors with TACI and BCMA (92). BLyS and APRIL act as survival macrophages, lymphokine-activated killer (LAK), natural factors for malignant myeloma cells cultured within the bone killer (NK) cells and T-lymphocytes (111). This and other marrow microenvironment and for B-cells of patients with antibody cytokine fusion proteins are presently under clinical Waldenströms macroglobulinemia (WM) (93-96). investigation (112-114). Atacicept is a fusion protein composed of the ECD of TACI and the Fc portion of human IgG1, resulting in Antibody-MHC Class I (peptide) Fusion Proteins neutralization of BLyS and APRIL (97). In vitro studies have demonstrated that Atacicept exerts a negative effect on Passive immunization of patients with cancer with proliferation and a pro-apoptotic effect on B-cells from monoclonal antibodies has resulted in a proven record of patients with lymphoma, multiple myeloma and WM and can therapeutic benefit (107, 115, 116). Another approach is also block the biological activity of heterodimeric complexes immunization of patients with cancer with peptides of this receptor family (97, 98). Atacicept is currently being recognized by T-lymphocytes and adoptive transfer therapies clinically investigated in phase I studies in patients with with tumor-reactive selected T-cells directed against tumor relapsed and refractory B-cell non-Hodgkin’s lymphoma antigens (117). These approaches are based on the (NHL), multiple myeloma and WM (99,100). Atacicept is elimination of tumor cells presenting MHC/peptide currently being also evaluated in several phase I to III studies complexes by cytotoxic T-cells. An important pitfall of this in patients with autoimmune diseases (101-103). approach is the inability to select tumor cell populations with loss of MHC expression, or dysfunctions in the MHC Antibody Cytokine Fusion Proteins peptide presentation pathway (118). Therefore, recombinant proteins have been designed, A promising strategy for the treatment of cancer is the which combine an antibody-based targeting module with a recruitment of immune effector cells to the tumor for fused β2 microglobulin MHC module. In some cases, the induction of an antitumor response. Systemic administration specific peptide binding to MHC has also been fused to the of IL2, granulocyte-macrophage colony stimulating factor designed proteins, as described above (119-122) (Figure 1D (GM-CSF), or IL12 were shown to increase the and 3E). The specific peptide can be derived from a tumor immunogenicity of tumors and their elimination, based on the or viral antigen. Proof-of-concept was demonstrated with a underlying immune response (104). However, severe side- single HLA-A2 molecule genetically fused to the variable effects have been observed, since systemic administration domains of antibody directed against the α subunit of the does not correspond to their physiological mode of action IL2R. Tumor cells coated with this fusion protein were (105). In order to increase the therapeutic index, cytokines susceptible to lysis by HLA-A2 restricted melanoma gp100 were genetically fused to antibody-relating targeting modules peptide specific cytotoxic T-cells (CTLs) (121). scab-sc directed against tumor antigens (106, 107) (Figure 1 A-C). HLA-A2 complexes targeting the α-subunit of the IL2R or The half-life of cytokines is significantly improved in human mesothelin with specificity for melanoma antibody cytokine fusion proteins. Pre-clinical in vivo efficacy differentiation antigen gp100 or an Epstein Barr virus data are summarized in (106, 107). In addition to their (EBV)-derived peptide epitope were evaluated in vivo after

366 Weidle et al: Genetically Engineered Fusion Proteins (Review) refolding and loading with the corresponding peptide. In vivo TCR/IL2), consisting of IL2 genetically-fused to soluble sc efficacy was demonstrated after intratumoral (i.t.) or HLA-A2-restricted TCR recognizing a peptide derived from intravenous (i.v.) injection of human CTLs (121). Another p53, which is overexpressed in tumors, was constructed. In example involved an antibody scFv fragment derived from this context, IL2 can be delivered directly to the tumor with EGFR antibody C225 fused to monomeric sc-HLA-A2 minimized toxicity. With human melanoma A375 xenografts complexes containing immunodominant tumor-related or (p53+/HLA-2.1+) in nude mice better than equivalent viral epitopes (122). The corresponding fusion protein antitumor doses of IL2 was noted. Antitumor activity was mediated CTL-dependent lysis of EGFR-expressing tumor mediated by NK cell activation and tumor infiltration. Tumor cells regardless of the expression of self-peptide MHC growth inhibition was observed in p53+/HLA-A2+, not in complexes. In vivo efficacy in corresponding xenografts was p53+/HLA-A2– tumors. Furthermore it was shown that the shown after i.t. and i.v. administration of EBV-specific CTLs. ECD of a TCR can be genetically fused to anti-CD3 A potential problem associated with the MHC peptide antibodies for recruitment of T-cells (131). Soluble high- complexes is the stability of the complex since the peptide affinity TCR, specific for HLA-A2-presented melanoma- can dissociate from the MHC binding groove. Therefore, associated epitope gp100 281-288 was fused to anti-CD3 sc N-terminal peptides were fused with sc-HLA-A2 molecules Fv domain. The activation of a polyclonal T-cell response, and C-terminal scFv of an anti-IL2R α subunit-specific when targeted to melanoma cell presenting the gp100 derived antibody. It was shown that the fusion protein induced HLA- epitope, was reported. Pico-molar (pM) activity was found A2- restricted, specific CTL-mediated lysis after binding to in a panel of tumor cells. Inhibition of tumor growth was antigen-positive tumor cells (122). In comparison to the use observed after co-injection of the fusion protein with human of bi-specific antibodies, the approach, as described, induces peripheral blood monocuclear cells. Phase I studies have a monoclonal instead of a polyclonal T-cell response. It been performed (132). should be stressed out that in none of the described fusion proteins is an Fc moiety incorporated. As outlined, Fc Concluding Remarks regions can impact pharmacokinetic properties and are mediators of antibody-based effector functions. From a technical point of view, genetically engineered fusion proteins confer improved pharmacokinetic characteristics on NK Cell Receptor Ligand Fusion Proteins decoy receptors and cytokines. This allows for less frequent and optimized dosing of these agents in comparison to their NK cells can eliminate tumor cells in the absence of MHC non-fused counterparts (134). Potential immunogenicity, due restriction, making use of multiple activating receptors (123). to the newly-generated junctions is a theoretical concern, but One of the activating receptors is NKG2D (124, 125). does not match the clinical observations. Tumor-targeting is Ligands in human are MHC class I chain-related protein A/B dependent on the tumor specificity of the antigen that the (MICA/B) and retinoic acid early-transcript (RAET1), also antibody-based molecule is directed against. In the case of called ULBPs (UL-16 binding proteins). NK2G2D ligands cytokines such as IL2 and TNFα, a significant improvement are preferentially overexpressed in tumor cells and tumor- of the therapeutic index was observed upon administration associated suppressor cells. It was shown that a fusion of the fusion proteins. protein between scFv anti-CD3 and the ECD of NKG2D Sequestration of Fc-based fusion proteins by interaction binds to NKG2D ligand-positive tumor cells and activates T- with Fc receptors also does not seem to be a problem. The cells via scFv anti-CD3 (126). Administration of scFv- maintainance of the cognate binding affinity of the ECD for NKG2D promoted survival in a murine lymphoma model. It its ligands must be investigated case-by-case and if was shown that an immunological memory response was necessary, can be improved by mutagenesis (91). generated in these mice. From a conceptional point of view, through creation of ECD-based fusion proteins by incorporation of ligand- TCR Fusion Proteins binding modules from different receptors, more potent decoy receptors can be generated in comparison to ECD-based Many tumor-related antigens are located intracellularly and fusion proteins derived from a single receptor. The specificity cannot be targeted with antibodies. Cancer testis antigens are of the antitumoral response mediated by immune effector one class of such targets among others (127, 128). Peptides cells is largely triggered by the specificity of the target derived from these targets are presented as T-cell epitopes molecule, its expression levels on tumor cells, its continuous complexed with MHC molecules and induce a cytotoxic expression and its tumor penetration. Down-modulation of T-cell response. The concept of a TCR-derived fusion protein the corresponding antigens targeted by the appropriate fusion for recruitment of immune effector cells to the tumor has proteins is a general mechanism of resistance. In the case of been described (129, 130). A cytokine fusion protein (264 sc fusion proteins targeting angiogenesis- or tumor-related

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